Citrus Seed Oils Efficacy against Larvae of Aedes aegypti.

Background
Dengue fever is a serious public health issue in Pakistan for many years. Globally plants have been reported to contain compounds with insecticidal properties. These properties have been demonstrated more recently on the larval stages of mosquitoes. Therefore, Citrus cultivar seeds were evaluated for larvicidal potential against the primary dengue vector Aedes aegypti.


Methods
Extraction of oil was done by a steam distillation method and oils were evaluated according to WHO guidelines for larvicides 2005 for evaluation of insecticidal properties of citrus seed extracts against mosquito larvae.


Result
Among the Citrus cultivar seed oil, rough lemon (Citrus jambhiri) had the lowest LC50 value (200.79ppm), while musambi (C. sinensis var musambi) had the highest LC50 value (457.30ppm) after 24 h of exposure.


Conclusion
Citrus cultivars have some larvicidal potential but C. jambhiri had the greatest potential against A. aegypti larvae. Further small-scale field trials using the extracts of C. jambhiri will be conducted to determine operational feasibility.


Introduction
Dengue fever (DF) and dengue hemorrhagic fever (DHF) are serious public health concerns in many developing countries, including Pakistan. Over 2.5 billion people over 40% of the world's population are now at risk from dengue. WHO currently estimates 50-100 million dengue infections occurred worldwide every year (1).
In Pakistan, the first case of DHF was observed in Karachi in 1994, (2) and 11,024 confirmed cases of DF including 40 deaths were reported in 2010 (3). In 2011, a subsequent dengue outbreak occurred with 22,778 confirmed cases and 353 deaths (4). The majority of the cases and deaths occurred in Lahore, Punjab Province while there was also epidemic in Khyber Paktunkhua (KPK) during 2013 that resulted in 23 deaths Apart from the KPK province, sporadic cases have also been reported from the provinces of Punjab, Sindh, and Balochistan (5).
Unlike yellow fever, there is no vaccine for dengue fever. However Osorio et al. (6) is developing a vaccine, which is still in the preclinical stage. Therefore, the only current effective approach to control dengue is through vector control. This is done mainly through integrated vector management programmes using insecticides, environmental management and public awareness (7,8). Due to the toxic effects and resistance to synthetic insecticides (9), vector control managers are facing problems in controlling mosquitoes. Therefore, it is necessary to develop safe alternative insecticides, which require minimum care (10).
In the view of an increased interest in the development of plant-based insecticides as alternatives to synthetic insecticides, this study was conducted to assess the larvicidal potential of citrus cultivars against the dengue vector A. aegypti.

Collection and Rearing of mosquitoes
Aedes aegypti larvae were collected from old tire shops of Sargodha (32.0836° N, 72.6711° E) and, reared in 3-inch deep steel trays, and were reared in the insectary of biosystematics Lab University of Agriculture Faisalabad-Pakistan. Larvae were fed on Tetra-min® fish feed until adult emergence (17). Adults were maintained in well-aerated cages (70× 35× 35cm), where the mosquitoes were provided with cotton wicks soaked in 10% sucrose solution. Females were also fed blood from white mice every alternate day (18). A Petri dish with landing pad was provided to lay their eggs. The population was maintained at set conditions of 27±2 o C, 75±5% RH and L14:D10 photoperiod.

Extraction of oil
The seeds of citrus cultivars were washed to remove the pulp, oven dried for 48 hours at 60 ˚C and later ground in an electric grinder. A thimble was used to hold the grounded material and kept in an extraction tube of Soxhelt apparatus with extractor ID 38mm, extractor volume 85ml and flask volume 250ml (19) for the extraction of oil by steam distillation method using Diethyl-ether as solvent (250ml/ 20g sample). The cyclic time of extraction for each sample was 4-5h.

Bioassay
Six different concentrations increasing by 100ppm from 300-800ppm of extracted oils were used with three replicates for each treatment, each replicate containing 200ml of the oil solution placed in 250ml glass beakers. Batches of 30 late 3 rd and early 4 th instar larvae of the A. aegypti were placed in each beaker (20). Control beakers were treated with diethyl-ether only. The experiment was conducted using Completely Randomized Design (CRD) under lab conditions at 27±2 ˚C and 70±5% relative humidity.

Data analysis
Abbot's formula (21) was used to correct for mortality, and this data was analyzed by probit analysis (22) using Minitab ® Statistical Software (23) software to create a dose mortality regression line. In the control treatment, if mortality rates were between 5% to 20% then percent mortality was corrected by Abbot's formula as follows: % corrected mortality= % observed mortality-% control mortality × 100 100-% control mortality

Results
The efficacy of citrus seed oils against the late 3 rd and early 4 th instar larvae of A. aegypti was expressed in terms of LC50 as shown in Table 2

Discussion
Mosquito borne diseases are one of the most important public health problems in the developing countries like Pakistan. Use of synthetic insecticides are the best option to control mosquito larvae but resistance, environmental problems etc are the some concerned problems which can be overcome by the use of Plant essential oils and extracts as a part of Integrated Vector Management (IVM).
A variety of plants is well known to contain chemicals with bioactive potential (24) as deterrents and attractants (25). The ether oils of different citrus cultivars (Table 2) have been studied as natural insecticides against A. aegypti larvae instead of synthetic insecticides as they are reported resistant to the mosquitoes especially A. albopictus (26). Rough lemon (C. jambhiri) had the lowest LC50 value (200.79ppm) against A. aegypti larvae. This is well supported by the findings of Akram et al. (17)

Conclusion
Our results indicated that out of the 9 citrus seed oils, rough lemon (C. jambhiri) had good larvicidal potential against late 3 rd and early 4 th instar larvae of A. aegypti in terms of LC50. Further studies should be done to investigate the larvicidal potential of rough lemon oil as well as other seed oils for the control of Aedes mosquitoes under field conditions.